Modular proteins from the Drosophila sallimus (sls) gene and their expression in muscles with different extensibility

The passive elasticity of the sarcomere in striated muscle is determined by large modular proteins, such as titin in vertebrates. In insects, the function of titin is divided between two shorter proteins, projectin and sallimus (Sls), which are the products of different genes. The Drosophila sallimus (sls) gene codes for a protein of 2 MDa. The N-terminal half of the protein is largely made up of immunoglobulin domains and unique sequence; the C-terminal half has two stretches of sequence similar to the elastic PEVK region of titin, and at the end of the molecule there is a region of tandem Ig and fibronectin domains. We have investigated splicing pathways of the sls gene and identified isoforms expressed in different muscle types, and at different stages of Drosophila development. The 5’ half of sls codes for zormin and kettin; both proteins contain Ig domains and can be expressed as separate isoforms, or as larger proteins linked to sequence downstream. There are multiple splicing pathways between the kettin region of sls and sequence coding for the two PEVK regions. All the resulting protein isoforms have sequence derived from the 3’ end of the sls gene. Splicing of exons varies at different stages of development. Kettin RNA is predominant in the embryo, and longer transcripts are expressed in larva, pupa and adult. Sls isoforms in the indirect flight muscle (IFM) are zormin, kettin and Sls(700), in which sequence derived from the end of the gene is spliced to kettin RNA. Zormin is in both M-line and Z-disc. Kettin and Sls(700) extend from the Z-disc to the ends of the thick filaments, though, Sls(700) is only in the myofibril core. These shorter isoforms would contribute to the high stiffness of IFM. Other muscles in the thorax and legs have longer Sls isoforms with varying amounts of PEVK sequence; all span the I-band to the ends of the thick filaments. In muscles with longer Ibands, the proportion of PEVK sequence would determine the extensibility of the sarcomere. Alternative Sls isoforms could regulate the stiffness of the many fibre types in Droso phila muscles

Comparing Models of Evolution for Ordered and Disordered Proteins

Most models of protein evolution are based upon proteins that form relatively rigid 3D structures. A significant fraction of proteins, the so-called disordered proteins, do not form rigid 3D structures and sample a broad conformational ensemble. Disordered proteins do not typically maintain long-range interactions, so the constraints on their evolution should be different than ordered proteins. To test this hypothesis, we developed and compared models of evolution for disordered and ordered proteins. Substitution matrices were constructed using the sequences of putative homologs for sets of experimentally characterized disordered and ordered proteins. Separate matrices, at three levels of sequence similarity (>85%, 85–60%, and 60–40%), were inferred for each type of protein structure. The substitution matrices for disordered and ordered proteins differed significantly at each level of sequence similarity. The disordered matrices reflected a greater likelihood of evolutionary changes, relative to the ordered matrices, and these changes involved nonconservative substitutions. Glutamic acid and asparagine were interesting exceptions to this result. Important differences between the substitutions that are accepted in disordered proteins relative to ordered proteins were also identified. In general, disordered proteins have fewer evolutionary constraints than ordered proteins. However, some residues like tryptophan and tyrosine are highly conserved in disordered proteins. This is due to their important role in forming protein–protein interfaces. Finally, the amino acid frequencies for disordered proteins, computed during the development of the matrices, were compared with amino acid frequencies for different categories of secondary structure in ordered proteins. The highest correlations were observed between the amino acid frequencies in disordered proteins and the solvent-exposed loops and turns of ordered proteins, supporting an emerging structural model for disordered proteins

Genomes of trombidid mites reveal novel predicted allergens and laterally-transferred genes associated with secondary metabolism

Trombidid mites have a unique lifecycle in which only the larval stage is ectoparasitic. In the superfamily Trombiculoidea (“chiggers”), the larvae feed preferentially on vertebrates, including humans. Species in the genus Leptotrombidium are vectors of a potentially fatal bacterial infection, scrub typhus, which affects 1 million people annually. Moreover, chiggers can cause pruritic dermatitis (trombiculiasis) in humans and domesticated animals. In the Trombidioidea (velvet mites), the larvae feed on other arthropods and are potential biological control agents for agricultural pests. Here, we present the first trombidid mites genomes, obtained both for a chigger, Leptotrombidium deliense, and for a velvet mite, Dinothrombium tinctorium

The evolutionary origin of bilaterian smooth and striated myocytes

The dichotomy between smooth and striated myocytes is fundamental for bilaterian musculature, but its evolutionary origin is unsolved. In particular, interrelationships of visceral smooth muscles remain unclear. Absent in fly and nematode, they have not yet been characterized molecularly outside vertebrates. Here, we characterize expression profile, ultrastructure, contractility and innervation of the musculature in the marine annelid Platynereis dumerilii and identify smooth muscles around the midgut, hindgut and heart that resemble their vertebrate counterparts in molecular fingerprint, contraction speed and nervous control. Our data suggest that both visceral smooth and somatic striated myocytes were present in the protostome-deuterostome ancestor and that smooth myocytes later co-opted the striated contractile module repeatedly for example, in vertebrate heart evolution. During these smooth-to-striated myocyte conversions, the core regulatory complex of transcription factors conveying myocyte identity remained unchanged, reflecting a general principle in cell type evolutio

Nucleotide Sequences Responsible for the Thermal Inducibility of the <i>Drosophila</i> Small Heat-Shock Protein Genes in Monkey COS Cells

The promoter regions of the Drosophila melanogaster small heat-shock protein genes have been analysed in order to localize those sequences responsible for their heat-shock transcriptional inducibility. Different lengths of the 5′ DNA sequences of these four genes were each fused individually to the Herpes simplex virus thymidine kinase (HSV-tk) transcription unit. These hybrid genes were constructed in a simian virus 40 recombinant vector for transfection in permissive monkey COS cells and tested for their heat-shock inducibility. The hsp22/HSV-tk and hsp26/HSV-tk fusion genes were found to be heat-inducible at 43 °C, giving rise to correctly initiated transcripts, but transcriptionally quiescent at 37 °C (control temperature). The hsp23 and hsp27 fusion gene constructs are, however, not heat-shock-inducible; no transcripts being detectable from hsp27/HSV-tk constructs at either temperature and all hsp23/HSV-tk clones being faithfully but constitutively expressed at low levels at both temperatures. By testing a series of 5′ deletion mutants in hsp22/HSV-tk, a homologous sequence located adjacent to the TATA box in both the hsp22 and hsp26 genes was identified as being responsible for their heat-shock activation. This control element corresponds to the Pelham “consensus sequence”, previously described for the Drosophila hsp70 genes. The possible modes of transcriptional induction of all four genes are discussed

Nucleotide Sequence Analysis of the <i>Drosophila</i> Small Heat Shock Gene Cluster at Locus 67B

The four small heat shock protein genes of Drosophila melanogaster clustered at cytological locus 67B have been characterized by DNA sequencing. Over 6250 nucleotides, covering the 5′, protein-coding and 3′ regions of these genes have been determined together with their predicted amino acid sequences. Each gene possesses characteristic eukaryotic 5′ and 3′ sequence elements and a single uninterrupted protein-coding region. The four encoded polypeptides of 19,700, 20,600, 23,000 and 23,600 Mr share a homologous stretch of 108 amino acid residues, representing 51 to 62% of their lengths. This region is flanked by sequences of dissimilar length and amino acid composition, located mainly at the amino-terminal end, but also at the extreme carboxyl termini of these proteins. The first 14 amino acids exhibit a small degree of homology, both amongst themselves and with some signal peptides and a transmembrane protein. Investigation of the hydrophilic/hydrophobic characteristics of the (four polypeptides revealed, within the conserved 108 amino acid stretch, the presence of an α-helical region of very prominent local hydrophilicity, which probably represents a surface structural domain common to each protein. Sequence analysis with respect to transcription initiation and termination and possible regulatory signals is discussed together with some structural predictions for the four proteins

Locus 67B of <i>Drosophila melanogaster</i> contains seven, not four, closely related heat shock genes

The four small hsp genes of Drosophila melanogaster as well as three genes regulated during development (genes 1, 2 and 3) are localized at the chromosomal locus 67B. The four small hsp genes share strong sequence homologies between themselves which were detected here by cross-hybridization. Under the same stringency conditions, each of the genes 1, 2 and 3 hybridize to some of the small hsp genes. By DNA sequencing of gene 1, the homology was localized within the same two regions already conserved between the small hsp genes: a central region of 83 amino acids, homologous with the mammalian alpha crystallin and the first 15 N-terminal amino acids. The transcriptional inducibility of the genes 1, 2 and 3 was also compared with that of the four small hsp genes during various stages of Drosophila development at either the normal growth temperature or after a heat shock. We confirm previous reports on the developmental patterns of all seven genes and find moreover that genes 1, 2 and 3 are heat-shock inducible at any of the stages tested. We conclude that genes 1, 2 and 3 are also heat shock genes. Therefore, the locus 67B contains seven, not four, small heat shock genes

Assembly of the giant protein projectin during myofibrillogenesis in Drosophila indirect flight muscles.

BACKGROUND: Projectin is a giant modular protein of Drosophila muscles and a key component of the elastic connecting filaments (C-filaments), which are involved in stretch activation in insect Indirect Flight Muscles. It is comparable in its structure to titin, which has been implicated as a scaffold during vertebrate myofibrillogenesis. METHODS: We performed immunofluorescence studies on Drosophila pupal tissue squashes and isolated myofibrils to identify the pattern of appearance and assembly for projectin and several other myofibrillar proteins, using both wild type and mutant fly stocks. RESULTS AND CONCLUSIONS: In the first step of assembly, projectin immunolocalization appears as random aggregates colocalizing with alpha-actinin, kettin and Z(210), as well as, F-actin. In the second step of assembly, all these proteins become localized within discrete bands, leading ultimately to the regularly spaced I-Z-I regions of myofibrils. This assembly process is not affected in myosin heavy chain mutants, indicating that the anchoring of projectin to the thick filament is not essential for the assembly of projectin into the developing myofibrils. In the actin null mutation, KM88, the early step involving the formation of the aggregates takes place despite the absence of the thin filaments. All tested Z-band proteins including projectin are present and are colocalized over the aggregates. This supports the idea that interactions of projectin with other Z-band associated proteins are sufficient for its initial assembly into the forming myofibrils. In KM88, though, mature Z-bands never form and projectin I-Z-I localization is lost at a later stage during pupal development. In contrast, treatment of adult myofibrils with calpain, which removes the Z-bands, does not lead to the release of projectin. This suggests that after the initial assembly with the Z-bands, projectin also establishes additional anchoring points along the thick and/or thin filaments. In conclusion, during pupation the initial assembly of projectin into the developing myofibril relies on early association with Z-band proteins, but in the mature myofibrils, projectin is also held in position by interactions with the thick and/or the thin filaments